Total error profiling of a proinsulin time-resolved fluorescence immunoassay

Area of Science:

• Biochemistry
• Analytical Chemistry
• Assay Development

Background:

• Enzyme-linked immunosorbent assay (ELISA) is a common method for proinsulin (PI) quantification.
• Time-resolved fluorescence immunoassay (TRFIA) offers potential advantages in sensitivity and dynamic range.
• Conversion of existing assays requires rigorous validation to ensure accuracy and reliability.

Purpose of the Study:

• To evaluate the conversion of a proinsulin (PI) enzyme-linked immunosorbent assay (ELISA) to a time-resolved fluorescence immunoassay (TRFIA).
• To assess the performance of the TRFIA assay using established bioanalytical acceptance criteria.
• To identify potential limitations or interferences in the converted TRFIA assay.

Main Methods:

• Total error profiling was employed to evaluate the assay conversion.
• The Ligand Binding Assay Bioanalytical Focus Group (LBABFG) of the American Association of Pharmaceutical Scientists (AAPS) proposed formula and acceptance criteria were applied.
• Assay performance was compared between the original ELISA and the developed TRFIA.

Main Results:

• The TRFIA assay demonstrated a wider dynamic range ([0.5-240] pmol/L) compared to the ELISA ([0.7-98] pmol/L).
• High concentrations of C-peptide (>7000 pmol/L) in samples caused interference in the TRFIA.
• This C-peptide interference limited the expected dynamic range enlargement of the TRFIA assay.

Conclusions:

• The conversion of PI ELISA to TRFIA is feasible but requires careful consideration of potential interferences.
• C-peptide interference at high concentrations is a critical factor limiting the TRFIA's dynamic range.
• Further optimization may be needed to mitigate C-peptide interference for accurate PI measurement using TRFIA.